Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.

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Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

Radiotherapy is one of the most common countermeasures for treating a wide range of tumors. However, the radioresistance of cancer cells is still a major limitation for radiotherapy applications. Efforts are continuously ongoing to explore sensitizing targets and develop radiosensitizers for improving the outcomes of radiotherapy. DNA double-strand breaks are the most lethal lesions induced by ionizing radiation and can trigger a series of cellular DNA damage responses (DDRs), including those helping cells recover from radiation injuries, such as the activation of DNA damage sensing and early transduction pathways, cell cycle arrest, and DNA repair. Obviously, these protective DDRs confer tumor radioresistance. Targeting DDR signaling pathways has become an attractive strategy for overcoming tumor radioresistance, and some important advances and breakthroughs have already been achieved in recent years. On the basis of comprehensively reviewing the DDR signal pathways, we provide an update on the novel and promising druggable targets emerging from DDR pathways that can be exploited for radiosensitization. We further discuss recent advances identified from preclinical studies, current clinical trials, and clinical application of chemical inhibitors targeting key DDR proteins, including DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), ATM/ATR (ataxia–telangiectasia mutated and Rad3-related), the MRN (MRE11-RAD50-NBS1) complex, the PARP (poly[ADP-ribose] polymerase) family, MDC1, Wee1, LIG4 (ligase IV), CDK1, BRCA1 (BRCA1 C terminal), CHK1, and HIF-1 (hypoxia-inducible factor-1). Challenges for ionizing radiation-induced signal transduction and targeted therapy are also discussed based on recent achievements in the biological field of radiotherapy.

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DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer.

Light controls important physiological and morphological responses in fungi. Fungi can sense near-ultraviolet, blue, green, red and far-red light using up to 11 photoreceptors and signalling cascades to control a large proportion of the genome and thereby adapt to environmental conditions. The blue-light photoreceptor functions directly as a transcriptional regulator in the nucleus, whereas the red-light-sensing and far-red-light-sensing phytochrome induces a signalling pathway to transduce the signal from the cytoplasm to the nucleus. Green light can be sensed by retinal-binding proteins, known as opsins, but the signalling mechanisms are not well understood. In this Review, we discuss light signalling processes in fungi, their signalling cascades and recent insights into the integration of light signalling pathways with other regulatory circuits in fungal cells.

Light sensing and responses in fungi.

How light affects the life of Botrytis

Research on the insect pathogenic filamentous fungus, Beauveria bassiana has witnessed significant growth in recent years from mainly physiological studies related to its insect biological control potential, to addressing fundamental questions regarding the underlying molecular mechanisms of fungal development and virulence. This has been in part due to a confluence of robust genetic tools and genomic resources for the fungus, and recognition of expanded ecological interactions with which the fungus engages. Beauveria bassiana is a broad host range insect pathogen that has the ability to form intimate symbiotic relationships with plants. Indeed, there is an increasing realization that the latter may be the predominant environmental interaction in which the fungus participates, and that insect parasitism may be an opportunist lifestyle evolved due to the carbon- and nitrogen-rich resources present in insect bodies. Here, we will review progress on the molecular genetics of B. bassiana, which has largely been directed toward identifying genetic pathways involved in stress response and virulence assumed to have practical applications in improving the insect control potential of the fungus. Important strides have also been made in understanding aspects of B. bassiana development. Finally, although increasingly apparent in a number of studies, there is a need for progressing beyond phenotypic mutant characterization to sufficiently investigate the molecular mechanisms underlying B. bassiana's unique and diverse lifestyles as saprophyte, insect pathogen, and plant mutualist.

Molecular Genetics of Beauveria bassiana Infection of Insects

Summary

Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here we show that Wee1 and Cdc25 can balance cell cycle-required cyclin-dependent kinase 1 (Cdk1) activity in Beauveria bassiana. The Cdk1 phosporylation signal was strong in Δcdc25 but very weak in Δwee1 and absent in Δwee1Δcdc25. Consequently, cell cycles, septation patterns and many septation-dependent gene transcripts of these mutants were reversely changed. Hyphal cells were short in Δwee1, slender in Δcdc25 and short and swollen in Δwee1Δcdc25. Conidiation was most defective in Δwee1, followed by Δcdc25. Their conidia and yeast-like blastospores also altered antagonistically in both size and complexity, accompanied with abnormally branched germlings in Δwee1 and Δwee1Δcdc25. Conidial thermotolerance and UV-B resistance decreased much more in Δwee1Δcdc25 than in Δwee1 but significantly increased in Δcdc25. The double deletion and the point mutation Cdk1T14A/P15F for inhibitory phosphorylation caused most defective virulence, followed by wee1 deletion. All the changes were restored by ectopic gene complementation. Virulence changes in all the mutants and control strains were highly correlated to those in blastospore size or complexity. Taken together, Wee1 and Cdc25 control cell cycle, morphogenesis, asexual development, stress tolerance and virulence of B. bassiana by balancing the Cdk1 activity.

Wee1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle‐required cyclin‐dependent kinase 1 activity

Three α-1,2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of Beauveria bassiana.

Summary

Phytochromes (Phy) in filamentous fungi are Group VIII histidine kinases that share a unique N-terminal photosensory core, but their functions are largely unknown. Here we show that Beauveria bassiana Phy (Bbphy) is functionally vital for growth, conidiation and multistress tolerance of the fungal entomopathogen lacking sexual stage. Colony growth of ΔBbphy was significantly slower in a nutrition-rich medium but faster in several minimal media. Conidial yield of ΔBbphy in the rich medium increased at the fitted rate of 3.4 × 107 conidia h−1 white light in the light/dark cycles of 0:24 to 16:8 h, decreased greatly in the short-, long- and full-day cycles of red/far-red light, but was unaffected under full-day blue light. Moreover, ΔBbphy showed higher osmosensitivity, increased antioxidant capability, and decreased conidial thermotolerance and UV-B resistance, accompanied with downregulation of Hog1 phosphorylation and of four Hog1-related genes under osmotic stress, and upregulation of five superoxide dismutases and four catalases under oxidative stress. All the changes were restored by the gene complementation. Taken together, Bbphy controls conidiation by responding to daylight length and red/far-red light and regulates multistress responses perhaps because of an involvement in Hog1 pathway. Our findings highlight diverse functions of Bbphy in B. bassiana.

Phytochrome controls conidiation in response to red/far-red light and daylight length and regulates multistress tolerance in Beauveria bassiana.

Gcn5 is a core histone acetyltransferase that catalyzes histone H3 acetylation on N-terminal lysine residues in yeasts and was reported to catalyze H3K9/K14 acetylation required for activating asexual development in Aspergillus. Here, we report a localization of Gcn5 ortholog in the nucleus and cytoplasm of Beauveria bassiana, a fungal insect pathogen. Deletion of gcn5 led to hypoacetylated H3 at K9/14/18/27 and 97% reduction in conidiation capacity as well as severe defects in colony growth and conidial thermotolerance. Two master conidiation genes, namely brlA and abaA, were transcriptionally repressed to undetectable level in Δgcn5, but sharply upregulated in wild-type, at the beginning time of conidiation. Based on chromatin immunoprecipitation, both DNA and acetylation levels of the distal and proximal fragments of the brlA promoter bound by acetylated H3K14 alone were upregulated in wild-type, but not in Δgcn5, at the mentioned time. In Δgcn5, normal cuticle infection was abolished while virulence through cuticle-bypassing infection was greatly attenuated, accompanied by drastically reduced activities of putative cuticle-degrading enzymes, retarded dimorphic transition and transcriptional repression of associated genes. These findings unveil a novel mechanism by which Gcn5 activates asexual development pathway by acetylating H3K14 and regulates the virulence-related cellular events in B. bassiana.

Gcn5-dependent histone H3 acetylation and gene activity is required for the asexual development and virulence of Beauveria bassiana.

Summary

Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2/M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

Juan-Juan Wang

Papers

Wee1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle‐required cyclin‐dependent kinase 1 activity

Three α-1,2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of Beauveria bassiana.

Phytochrome controls conidiation in response to red/far-red light and daylight length and regulates multistress tolerance in Beauveria bassiana.

Gcn5-dependent histone H3 acetylation and gene activity is required for the asexual development and virulence of Beauveria bassiana.

Unveiling equal importance of two 14‐3‐3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen